This invention is directed to recombinant DNase B derived from the pathogenic bacterium Streptococcus pyogenes, methods for its production, and methods for its use.
Despite advances in the prevention and treatment of bacterial infection, a number of bacterial pathogens remain serious problems in medical practice and continue to cause severe, even fatal disease. One of these pathogens is S. pyogenes. Among the diseases caused by S. pyogenes are streptococcal pharyngitis (xe2x80x9cstrep throatxe2x80x9d), scarlet fever, and their suppurative complications, including cervical adenitis, otitis media, mastoiditis, peritonsillar abscesses, meningitis, pneumonitis, pneumonia, puerperal sepsis, cellulitis of the skin, impetigo, lymphangitis, erysipelas, acute glomerulonephritis, and rheumatic fever.
Such infections often occur in hospitals (nosocomial infection), particularly in patients whose normal immune system functioning is suppressed. The latter category includes patients with AIDS, patients taking immunosuppressive drugs for cancer or to prevent transplant rejection, and patients having poor circulation, e.g., patients with diabetes.
Because these diseases require rapid and effective treatment to eradicate the suppurative lesions and prevent sequelae caused by immunological reactions to persisting suppurative lesions, prompt diagnosis of the presence of S. pyogenes is essential in patients in whom such infections are suspected. Failure to diagnose S. pyogenes promptly can greatly complicate treatment or even make it impossible.
Although detection methods for S. pyogenes are currently available, these methods have defects, particularly in clinical applications.
Among the methods of detection of S. pyogenes is the detection of the presence of antibodies against DNase B, a DNA-degrading enzyme produced by S. pyogenes. This enzyme, which is excreted from S. pyogenes during infection, initiates development of substantial titers of antibody in patients who go on to develop acute rheumatic fever and acute glomerulonephritis.
Although other serum-based diagnostic tests for these rheumatic fever and glomerulonephritis are available, including the detection of antibodies to streptolysin O, and to hyaluronidase, assays for anti-DNase B antibodies offer certain advantages, because DNase B is found among nearly all strains of group A beta-hemolytic streptococci, and because high DNase B titers are found in patients with infections of the skin and pharynx.
Although a number of commercially-available tests exist for the assay of anti-DNase B antibody, these tests have defects. As indicated above, an improved test is greatly needed.
The commercially-available tests fall into three categories: (1) a DNase B inhibition-based assay using the ability of the antibody to inhibit enzymatic activity; (2) a latex agglutination assay for antibody against a variety of S. pyogenes antigens; and (3) a turbidimetric inhibition assay. ELISA assays have also been used in the research laboratory, but, as detailed below, they have not yet proven suitable for routine clinical application.
The DNase B inhibition assay is very slow, and typically requires about 4-8 hours to perform. Thus, in situations in which confirmation of anti-DNase B antibody is required rapidly so the treatment can be started as soon as possible should the presence of S. pyogenes be confirmed, the enzyme inhibition assay is not particularly useful.
The latex agglutination assay is designed to detect antibodies to five S. pyogenes antigens. However, test results indicate poor agreement between the latex agglutination assay and a specific anti-DNase B tests. In one study, G. C. Klein and W. L. Jones, xe2x80x9cComparison of the Streptozyme Test with the Antistreptolysin O, Antideoxyribonuclease B, and Antihyaluronidase Tests,xe2x80x9d App. Microbiol. 21:257-259 (1971), 12 out of 80 patients that tested negatively in the latex agglutination assay were, in fact, positive for anti-DNase B antibody. This high level of false negative results means that the test is undesirable for clinical use.
The turbidimetric inhibition assay depends on the inhibition of agglutination of latex particles coated with anti-DNase B antibody by a limiting quantity of a crude preparation of DNase B in the presence of serum containing anti-DNase B antibody, which competes for the antibody on the latex particles. This assay, which is described in U.S. Pat. No. 5,055,395, incorporated herein by this reference, is relatively insensitive. Therefore, it is not suitable for use in the early stages of S. pyogenes infection, and it is precisely this period when accurate detection of the anti-DNase B antibody is most important. Additionally, the reagents used in the turbidimetric inhibition assay are difficult to manufacture.
ELISA-based assays for anti-DNase B antibody are reported in M. A. Gerber et al., xe2x80x9cEnzyme-Linked Immunosorbent Assay of Antibodies in Human Sera to Streptococcal DNase B,xe2x80x9d J. Lab. Clin. Med. 95:258-265 (1980). Although these assays have proven effective as research tools, their scale-up for commercial use, particularly in clinical practice, has been impractical. This is because such scale-up would require production and purification of the DNase B enzyme of Streptococcus pyogenes, which is, as detailed above, a serious pathogen. Not only would extremely costly containment methods be required for growth of this pathogenic bacterium in the quantity required to produce sufficient enzyme for commercialization of the ELISA assay, the media required for the growth of S. pyogenes is very complex and expensive. These concerns have seriously hampered development of a commercial version of the ELISA assay for anti-DNase B antibody.
Therefore, there exists a need for an improved, rapid, and specific assay for anti-DNase B antibody. Preferably, such an assay would be usable by a physician in his office and would require minimal equipment. This is because patients with diseases such as strep throat or scarlet fever typically see their family physician prior to hospitalization, and accurate diagnosis of S. pyogenes infection at that point would be preferable to a subsequent diagnosis made only when the patient has been hospitalized.
The development of such an improved assay is dependent on the availability of large quantities of DNase B enzyme itself. Therefore, there is also a need for a method for the production of S. pyogenes DNase B enzyme using a procedure that can be scaled up to produce commercial quantities of the enzyme without requiring complex, unwieldy, and expensive containment measures.
We have cloned and expressed the gene for S. pyogenes DNase B in Escherichia coli, allowing convenient and efficient production of the DNase B enzyme without requiring the growth of S. pyogenes. 
This cloning procedure results in substantially purified DNA encoding an amino acid sequence selected from the group consisting of the amino acid sequence of: (i) Streptococcus pyogenes DNase B enzyme as shown in FIG. 4 (SEQ ID NO:9), below, which enzyme includes at its amino terminus an arginine. (R) residue derived from a leader peptide and absent in the i0 natural DNase B enzyme; and (ii) a sequence encoding a functional equivalent of S. pyogenes DNase B enzyme, optionally including at least one residue of the leader peptide. The DNA is substantially free of DNA other than DNA encoding the S. pyogenes DNase B sequence of FIG. 4 (SEQ ID NO:9), DNA encoding a functional equivalent of S. pyogenes DNase B enzyme, and DNA encoding the leader peptide.
Preferably, the DNA further comprises a DNA sequence coding for a leader peptide fused to the amino terminus of S. pyogenes DNase B enzyme.
Most preferably, the DNA cloned is the DNA whose sequence is given in FIG. 3 (SEQ ID NO:7), including the DNA coding for the entire amino acid sequence of S. pyogenes DNase B enzyme and the leader peptide.
Another aspect of the invention is expression vectors for Streptococcus pyogenes DNase B enzyme comprising the DNA sequences described above operatively linked to at least one control sequence compatible with a suitable bacterial host cell. Preferably, the expression vector is a plasmid vector. Typically, the DNA encoding the Streptococcus pyogenes DNase B enzyme is linked to at least one sequence from bacteriophage xcex.
Another aspect of the invention is a bacterial host cell transformed, transfected, or infected with an expression vector according to the present invention in a manner allowing the transformed bacterial host cell to express the Streptococcus pyogenes DNase B encoded by the DNA incorporated within the expression vector in a detectable quantity. The expressed S. pyogenes DNase B can be either excreted or not excreted by the whole cell producing the enzyme, and can be in a soluble or an insoluble form.
Another aspect of the invention is substantially purified S. pyogenes DNAse B enzyme comprising a protein having the amino acid sequence of FIG. 4 (SEQ ID NO:9), or having an amino acid sequence substantially similar thereto.
Yet another aspect of the invention is a process for producing substantially purified Streptococcus pyogenes DNase B enzyme comprising:
(1) culturing the bacterial host cell transformed with an expression vector according to the present invention;
(2) using the cultured bacterial host cell to express the DNase B enzyme; and
(3) purifying the enzyme from the cultured bacterial host cell.
Another aspect of the invention is Streptococcus pyogenes DNase B enzyme fused at its amino terminus with a leader peptide, the leader peptide having the sequence M-N-L-L-G-S-R-R-V-F-S-K-K-C-R-L-V-K-F-S-M-V-A-L-V-S-A-T-M-A-V-T-T-V-T-L-E-N-T-A-L-A-R (SEQ ID NO:1).
Yet another aspect of the invention is a mutant of the protein whose amino acid sequence is shown in FIG. 4 (SEQ ID NO:9). In the mutant, at least one of the following mutations occurs:
(1) a deletion of one or more amino acids from the sequence of FIG. 4 (SEQ ID NO:9);
(2) an insertion of one or more naturally-occurring L-amino acids into the sequence of FIG. 4 (SEQ ID NO:9); and
(3) replacement of at least one of the amino acids of FIG. 4 (SEQ ID NO:1) with an alternative naturally occurring L-amino acid. The resulting mutant has reduced or increased DNase B activity or another altered property. In one preferred alternative, the mutant substantially retains the antigenic reactivity of natural S. pyogenes DNase B enzyme.
Yet another aspect of the invention is the translational or transcriptional fusion of all or part of the S. pyogenes DNase B gene or protein to another gene or protein, with the resulting genetic construction having some altered property. These properties can include: (1) high level RNA expression; (2) high level protein expression; (3) a second functional enzyme, receptor, or other active protein in the fusion; (4) the fusion of the DNase B to an affinity ligand; (5) the production of a higher molecular weight protein; and (6) increased immunoreactivity.
Still another aspect of the invention is substantially purified natural Streptococcus pyogenes DNase B enzyme substantially free of proteins other than Streptococcus DNase B enzyme and Streptococcus DNase B enzyme fused at its amino terminus with a leader peptide. The substantially purified protein is substantially free of mitogenic activity. The substantially purified enzyme can be further purified into two fractions, Fraction I and Fraction II, depending on isoelectric point (pI). Each fraction can be purified into a preparation substantially free of the other fraction.
A process according to the invention for preparing substantially purified natural S. pyogenes DNase B enzyme can comprise:
(1) absorption to and elution from diethylaminoethyl cellulose to produce a first eluate;
(2) chromatography of the first eluate on phenyl agarose to produce a second eluate;
(3) chromatography of the second eluate on heparin agarose to produce a third eluate; and
(4) chromatofocusing of the third eluate to produce substantially purified DNase B enzyme. Preferably, the process further comprises purification of the substantially purified DNase B by reverse-phase high-pressure liquid chromatography. The separation of Fractions I and II occurs at the chromatofocusing step as a consequence of the differing pI""s of the enzymes of the two fractions.
Yet another aspect of the invention is a single-stranded nucleic acid probe hybridizing with at least about 17 nucleotides of the DNA sequence coding for the amino-terminal 23 amino acids of the Streptococcus pyogenes DNAse B enzyme, not including any portion of the leader sequence thereof, with no greater than about a 30% mismatch.
A further aspect of the present invention includes portions of the DNA sequence of sufficient size and specificity to serve as primer sites for amplification reactions such as polymerase chain reaction (PCR), ligase chain reaction (LCR), RCR, or other DNA amplification reactions. The same portions of the DNA sequence of S. pyogenes B can also serve as specific probes for detection of homologous sequences without DNA amplification.
The substantially purified S. pyogenes DNase B can be used to generate antibodies specifically binding the DNase B by techniques well known in the art. The antibodies can be either polyclonal or monoclonal.
Another aspect of the invention is a method for detecting and/or determining anti-Streptococcus pyogenes DNase B antibody in a test sample. The method comprises the steps of:
(1) providing a test sample suspected of containing anti-Streptococcus pyogenes DNase B antibody;
(2) adding a quantity of Streptococcus pyogenes DNase B enzyme according to the present invention to the test sample, the quantity being sufficient to produce a detectable level of enzymatic activity in the absence of inhibition of the enzymatic activity by anti-DNase B antibody in the test sample; and
(3) determining a level of activity of DNase B enzyme in the test sample by performing an enzyme assay to detect and/or determine the anti-Streptococcus pyogenes antibody in the test sample.
An alternative method for detecting anti-DNase B antibody comprises the steps of:
(1) binding Streptococcus pyogenes DNase B enzyme according to the present invention to a solid support such as latex particles;
(2) reacting a test sample suspected of containing anti-Streptococcus pyogenes DNase B antibody with the Streptococcus pyogenes DNase B enzyme bound to the solid support to bind the antibody to the enzyme and thus to the solid support; and
(3) detecting the antibody bound to the solid support to detect and/or determine the antibody in the test sample.
This approach can be used for nephelometric, turbidimetric, agglutination, or ELISA methods of quantitation.
An alternative method for detecting S. pyogenes DNase B antibody comprises:
(1) preparing a buffered solution of DNase B; (2) reacting the buffered DNase B solution with a test sample suspected of containing anti-S. pyogenes DNase B antibody; and (3) detecting a reaction between the DNase B and the anti-DNase B antibody by observing and/or measuring a change in light absorption and/or light scattering in the solution.
Another alternative method for detecting anti-DNase B antibody is capillary electrophoresis.
Because the cloned sequence includes a promoter associated with the S. pyogenes DNase B, gene, yet another aspect of the invention is a method of using the promoter originally associated with the S. pyogenes DNase B gene to express a protein other than DNase B. This method comprises:
(1) separating the promoter originally associated with the S. pyogenes DNase B gene from the S. pyogenes DNase B gene;
(2) operatively linking the promoter with a structural gene for a S. pyogenes protein other than the gene for DNase B; and
(3) expressing the protein encoded by the structural gene.
The protein can be expressed in S. pyogenes, or in a prokaryote other than S. pyogenes. 
Another aspect of the invention is a substantially purified promoter sequence derived from the promoter sequence originally associated with S. pyogenes DNase B including therein a start site for transcription and sites homologous to the consensus xe2x88x9210 and xe2x88x9235 sites of bacterial promoters.
Yet another aspect of the present invention is the use of the leader peptide of DNase B with the sequence M-N-L-L-G-S-R-R-V-F-S-K-K-C-R-L-V-K-F-S-M-V-A-L-V-S-A-T-M-A-V-T-T-V-T-L-E-N-T-A-L-A-R (SEQ ID NO:1) to express a protein in a prokaryote. This aspect derives from the finding that when the entire cloned DNase B DNA segment, including the leader peptide, is expressed in Escherichia coli, the protein is excreted into the culture medium. A process for using the leader peptide to express a protein in a prokaryote comprises:
(1) fusing the DNA coding for the protein to DNA coding for the leader peptide so that the fused DNA codes for a recombinant protein with a single reading frame with the leader peptide being at the amino-terminus of the protein;
(2) introducing the fused DNA into the prokaryote; and
(3) expressing the fused DNA in the prokaryote so that the recombinant protein is produced in a recoverable quantity.
The prokaryote can be E. coli or a gram-positive bacterium such as a Staphylococcus, Streptococcus, or Streptomyces species.
Another aspect of the present invention is a method for immunizing a mammal against infection with S. pyogenes comprising administering a quantity of purified S. pyogenes DNase B enzyme according to the present invention to the mammal sufficient to stimulate production of antibodies specific for S. pyogenes DNase B.
Yet another aspect of the present invention is a method for treating cystic fibrosis in a patient with cystic fibrosis. The method comprises:
(1) generating an aerosol of a purified enzymatically active DNase B enzyme according to the present invention; and
(2) administering the aerosol to a patient with cystic fibrosis in a quantity sufficient to reduce lung fluid viscosity in the patient.